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View Article OnlineView Journal | View Issue

Normal saline-in

aCentre of Excellence for Biophotonics, Depa

Manipal Academy of Higher Education, Ma

santhosh.cls@manipal.edubDepartment of Immunohematology and Bloo

Manipal Academy of Higher Education, Man

Cite this: RSC Adv., 2019, 9, 7878

Received 7th December 2018Accepted 18th February 2019

DOI: 10.1039/c8ra10061f

rsc.li/rsc-advances

7878 | RSC Adv., 2019, 9, 7878–7884

duced deoxygenation of red bloodcells probed by optical tweezers combined with themicro-Raman technique

Jijo Lukose, a Mithun N,a Ganesh Mohan, b Shamee Shastry b

and Santhosh Chidangil *a

The use of normal saline for washing red blood cells and treating critically ill patients is a regular medical

practice in hospital settings. An optical tweezer in combination with Raman spectroscopy is an analytical

tool employed for the investigation of single cell dynamics, thus providing molecular fingerprint of the

cell by optically trapping the cell at a laser focus. In this study, the impact of normal saline on individual

human red blood cell was compared with that of blood plasma using Raman tweezers spectroscopy.

Major spectral variations in the marker frequencies at 1209 cm�1, 1222 cm�1, 1544 cm�1, and 1561 cm�1

of the Raman spectrum of the treated cells imply that the transition of hemoglobin to the deoxygenated

state occurs when 0.9% normal saline is used. This may result in serious implications in blood

transfusion. The results obtained from the principal component analysis also displayed clear

differentiation among the red blood cells diluted in normal saline and those diluted in plasma. In future

studies, efforts will be made to correlate the deoxygenation status of red blood cells with various human

disorders.

Introduction

The benet versus safety of normal saline in the medical eld isa subject of debate; in this regard, many researchers havereviewed the safety of using normal saline (0.9% NaCl) indifferent clinical settings1 since it is one of the most commonlyused solutions in the hospital settings. In transfusionmedicine,normal saline is used for cell washing and salvaging, inapheresis and for the resuscitation of patients with blood oruid loss. Washing of red blood cells (RBCs) is carried out using1 or 2 liters of sterile normal saline. This process is typicallyperformed to remove plasma proteins and glycerol from thefrozen RBC units. As the process of washing removes the anti-coagulants and preservative solution, the shelf life of the RBCproduct is reduced. As per the American Association of BloodBanking guidelines, the washed RBC unit can be stored at 1–6 �C for 24 hours.2 During apheresis, normal saline is used toprime the circuit and as a replacement uid in the therapeuticplasma exchange procedures. Moreover, it is used during intra-operative cell salvaging to wash the red blood cells. In a recentstudy, normal saline could induce higher levels of hemolysis ascompared to Plasma-Lyte A aer the cells were washed with it

rtment of Atomic and Molecular Physics,

nipal, Karnataka, India-576104. E-mail:

d Transfusion, Kasturba Medical College,

ipal, Karnataka, India-576104

and stored for a short term (24 h or less).3 Previously, low levelsof hemolysis were not a matter of concern as they were thoughtto be harmless and frequently present in transfused red cells;however, they may be injurious and need to be furtherresearched; this observation is very critical considering thateven low levels of hemolysis can trigger the probability of vitalorgan failure, vasculopathy, and predispose to nosocomialinfection. Infusion of normal saline in high volumes duringresuscitation and apheresis can cause hyperchloremic meta-bolic acidosis, which may impair the renal function.4 Normalsaline is also commonly used as an isotonic buffer for red bloodcells in research protocols. Some groups have also used it forspectroscopic investigation of live red blood cells.5

The emergence and major developments occurring in theinterdisciplinary “biophotonics” area have contributed to theorigin of many spectroscopic techniques specialized in theexamination of a plethora of biological/biochemical processes.The knowledge of various blood components, such as bloodcells, platelets and white blood cells, is of paramount impor-tance in recognizing different conditions of the human body.Raman spectroscopy is widely regarded as a highly reliableoptical tool for investigating biological systems.6,7 However, anytype of direct analysis of live cells suspended in a liquidmedium is highly restricted by the random movement of thesemicron-sized particles due to Brownian motion. This problemcan be tackled using optical tweezers, which capture a micro-scopic particle with the use of a tightly focused laser beam.Optical tweezers, developed by Arthur Ashkin, have been

This journal is © The Royal Society of Chemistry 2019

Fig. 1 Schematic of the Raman tweezers experimental setup; the insetshows RBC (a) before and (b) after trapping.

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gaining signicant attention from the scientic fraternity,especially from biophysicists, due to their ability to manipulateand trap a single cell.8,9 The use of optical tweezers in combi-nation with Raman spectroscopy has opened up new avenuesfor the inspection of live cells and their interactions.10 Basically,in the Raman tweezers system, a beam is used to trap a singlelive cell, and the same or another laser beam simultaneouslyexcites and generates Raman signals. Thus, the cell can be easilymanipulated for the required studies. The advantages of theRaman tweezers technique are its high specicity to identify thechemical moieties of an individual biological cell and real-timeidentication of the biochemical alterations in the presence ofexternal stimuli; moreover, minimal sample preparation andsample volume are required in this technique. In addition,since it is a label-free technique, the time-consuming uores-cent or radioactive labelling of the biological analytes is notrequired. Researchers have already applied this tool to obtainvital information regarding the biochemical alterations occur-ring in a single, live red blood cell due to external factors such astemperature, laser irradiation, silver nanoparticles, glucoseetc.11–14 Raman tweezers technique was also explored for moni-toring the oxygenation states of hemoglobin in red blood cellsand their relation with various human health disorders.15,16 Thistechnique has several applications in medicine as well as in cellbiology, diagnosing cell disorders by investigating the cellularmechanisms at the individual cell level.17–19

The comparison of normal saline with blood plasma asa medium for red blood cell studies is an important area ofresearch; however, the use of Raman tweezers to probe normalsaline-induced modications on a single RBC has not been re-ported to date. To the best of our knowledge, this is the rststudy describing the saline-induced spectral changes of redblood cells. This study presents the micro-Raman spectroscopiccomparison of a single, live red blood cell optically trapped andsuspended in two media: normal saline and human bloodplasma. Signicant alterations in the known oxygenationmarker bands reveal a transition from the oxy to the deoxy statewhen red blood cells are suspended in normal saline.

ExperimentalSample preparation

Sample collection was conducted by the Blood Bank, KasturbaMedical College, Manipal, India. The national guidelines onblood donor acceptance were followed to select the studysubjects.20 Informed consent was obtained from all subjects. Allthe donors in the study were males, and their age was between18 and 60 years. All the samples had a hemoglobin level above12.5 g dl�1. Moreover, infection screening tests for HIV, HBV,HCV, malaria and syphilis were found to be negative for thesamples. Fresh whole blood obtained from the volunteers wascentrifuged for 5 minutes at 3000 rpm to separate the packedred blood cells (PRBC). The study subjects did not consume anyethanol-containing products for 24 hours prior to sampling; inthis procedure, the packed red blood cells obtained from thevolunteers were highly diluted and suspended in blood plasmaor a normal saline solution for ten minutes prior to conducting

This journal is © The Royal Society of Chemistry 2019

the Raman measurements. AB blood group plasma obtainedfrom the volunteers selected based on the national guidelineswas used for the study.20 The dilution was made to avoidmultiple cell trapping due to laser spot. All Raman spectra wereacquired within two hours aer the blood was obtained anddiluted in the media solution. The power levels that are used toobtain the Raman spectra of the treated red blood cells in boththe saline and the plasma are well below the threshold for theoccurrence of photo-dissociation. In all the studies, measure-ment was performed using normal saline (0.9% NaCl obtainedfrom Infutec Healthcare Limited), and the PRBC suspension inblood plasma was used as a control. Permission was obtainedfrom the Institutional Ethics Committee, Kasturba MedicalCollege and Kasturba Hospital, on 17th January 2018 (IEC: 68/2018) before initiation of the study.

Instrumentation

The present study was carried out using a custom-built, single-beam Raman tweezers instrument (Fig. 1).21 A 785 nm laserbeam (Starbright Diode Laser, Torsana Laser Tech, Denmark)was used for trapping as well as excitation of the live red bloodcells diluted in isotonic media (0.9% NaCl medium or Plasma).An inverted microscope (Nikon Eclipse, Ti-U, Japan) with a highnumerical aperture (1.3 NA) and 100X oil immersion micro-scope objective (Nikon, Plan Fluor, Japan) was used to realizethe tight focusing of the laser beam. The resultant scatteredRaman signals were obtained using the same objective anddirected towards a spectrometer (Horiba Jobin Yvon iHR320with 1200 grooves per mm grating blazed at 750 nm) and liquidnitrogen-cooled CCD (Symphony CCD-1024 � 256-OPEN-1LS).Since the laser exposure time on an individual cell wasrestricted to 60 s and a low power of �7.5 mW was used, thechance of photo damage to the cell was little; thus, the cellremained intact, and the resultant Raman spectrum was valid.The spectra were never obtained on the same red blood cellrepeatedly to minimize the photo-damage. Baseline correctionas well as normalization of the Raman spectra aer smoothing

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were performed using MATLAB via the vector normalizationmethod. The image of RBC obtained before and aer trapping isshown in the inset of Fig. 1.

Table 1 Band assignments of RBCs that underwent major changes inthe presence of normal saline

Wavenumber(cm�1) Intensity variation Band assignment

565 Y n(Fe–O2)752 Y n151209 [ n5 + n181222 Y n13 or n421397 Y n201521 [ n381544 [ n111561 Y n21603 [ n(C]]C)vinyl1617 Y n(C]]C)vinyl1636 Y n10

Results and discussion

In this section, the changes observed for the optically trappedsingle RBC when the suspended medium is changed fromplasma to normal saline have been discussed using Ramanspectroscopy. The comparative spectra of the RBC diluted innormal saline and blood plasma are shown in Fig. 2, in whichthe major spectral variations are highlighted. The acquisitiontime for each spectrum was 60 s with two accumulations. Eachspectrum shown in the gure is an average of the spectra of 5different cells obtained from the same individual. To validatethe spectral changes, the experiment was repeated by obtainingthe blood from two more volunteers, and the correspondingspectra are shown in Fig. 2(b) and (c). The experiments for eachset of samples were performed in less than one hour.

Hemoglobin is the major molecular species in the cytoplasmof RBCs, and themajority of peaks present in the Raman spectraof the red blood cell originate from hemoglobin. Hemoglobin isresponsible for the transport of oxygen via binding of the hemegroups to oxygen. In general, this protein is described by a two-state model between two alternate structures: (1) a deoxygen-ated (deoxy), tense (T) structure and (2) an oxygenated (oxy),relaxed (R) structure. The form of hemoglobin that is bound tooxygen is termed as oxyhemoglobin, whereas the form that is

Fig. 2 Average Raman spectra of RBC diluted in normal saline and plasm

7880 | RSC Adv., 2019, 9, 7878–7884

not bound to oxygen is termed as deoxyhemoglobin.22 Ahemoglobin molecule consists of four polypeptide globin chainsubunits that comprise a heme group in each of it. This hemegroup is composed of a porphyrin ring that has an iron atom inits centre with six coordination sites; the four coordination sitesof iron are occupied by the porphyrin nitrogen, whereas the hsite is connected to the histidine residue in the globin chain.23

Once the sixth coordination site of iron binds with oxygen, theiron atom lies in the porphyrin plane and thus adopts a planarconguration in the oxygenated state.24 Once oxygen is removedfrom the heme, the iron atom is pulled out of the porphyrinplane towards the histidine residue; this results in a domed

a from three volunteers (a)–(c).

This journal is © The Royal Society of Chemistry 2019

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conguration for the deoxygenated form.24,25 The peaks dis-playing the major changes induced by normal saline are pre-sented in Table 1 along with their band assignments.21

Primarily, the oxygenation trend is veried via inspecting thespin-state marker region of heme, as shown in Fig. 3(a) and (b).The spin-state marker region (1500–1650 cm�1) is regarded asan important region of interest due to the presence of prom-inent Raman bands related to the oxygenation state of hemo-globin. The Raman signals appearing at 1544 cm�1, 1561 cm�1,1617 cm�1, and 1636 cm�1 are all contributed by the C–C bondsin the porphyrin ring, which rely on the spin states of the ironatom.26 As abovementioned, during the transition from the oxyto the deoxy state, the iron atom in the ferric low spin state isconverted to the ferrous form due to electron withdrawal; thisforces the displacement of the Fe atom 4 nm out of theporphyrin plane.25,27 This conformational change will beimmediately reected in the vibrational modes linked with theporphyrin ring, which can be tracked via the Raman spectra.The increase in the intensity of 1521 cm�1, 1544 cm�1 as well as1603 cm�1 accompanied by a decrease in the intensity of1561 cm�1 can be assigned to the presence of more number ofdeoxy hemes in the blood cell. In the case of normal saline, thepeaks present at 1617 cm�1 band and 1636 cm�1 (d(CɑCm)asym) band experienced a reduction in intensity; this could belinked with porphyrin doming.26

Furthermore, the oxygenation trends in the red blood cellsdiluted with saline and plasma were revaluated by monitoringthe bands present in the 1200–1230 cm�1 spectral region(Fig. 3(c)). These are the spectral regions assigned to themethine C–H deformations in the heme, which are readilyinuenced by the coordination between the porphyrin ring andthe iron atom.28 Any conformational change that occurs in

Fig. 3 Average Raman spectra of RBCs diluted in normal saline and plasm(c) the methine deformation region, (d) the FeO2 stretch, and (e) the po

This journal is © The Royal Society of Chemistry 2019

hemoglobin leads to an immediate variation in the deformationangle of the C–H vibrations due to the proximity of thesevibrations to the protein subunits.24 This subsequently resultsin the Raman shi of the corresponding bands due to thetransition from the oxygenated to the deoxygenated state. In thecase of normal saline, a gain in the intensity of 1209 cm�1 peakaccompanied by a loss in the intensity of 1222 cm�1 peak wasobserved. Earlier studies have correlated this intensity shiwiththe oxy-deoxy state transition due to the conformationalchanges occurring in heme.29 To conrm the saline-induceddeoxygenation, the well-known spectral region correspondingto heme-bound oxygen and the FeO2 stretch present at 565 cm�1

(Fig. 3(d)) are considered. In the case of normal saline, the dropin the O2 concentration is clearly visible from the decline in theintensity of the band at 565 cm�1.

The overlaid plot of the spectral region centered at 752 cm�1

is shown in Fig. 3(e). This peak corresponds to the C–N–Cvibration in the porphyrin ring, which is generally used as anindicator for the number of intact hemoglobin moleculespresent in red blood cells.5 In general, the intensity of theporphyrin breathing mode is regarded as a marker for thevitality of RBCs. A decrease in the intensity of the band isobserved for normal saline as compared to the case of bloodplasma. This variation also questions the stability of RBCs innormal saline for hospital applications. Similar to the spin-statemarker region, the pyrrole ring stretching region (1300–1400 cm�1) is highly sensitive to the oxidation state of ironpresent in the porphyrin ring. A decrease in the intensity of theband at 1397 cm�1, which represents the pyrrole quarter ringstretching, is found in the case of the normal saline samples(Fig. 2(a)). In addition, the band assigned to the pyrrole half ringstretching, which appears at 1375 cm�1 in the oxygenated state,

a where spectral variations are dominant (a), (b) the spin marker region,rphyrin breathing mode.

RSC Adv., 2019, 9, 7878–7884 | 7881

Fig. 5 Oxygen purging of RBC in normal saline.

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is shied to 1370 cm�1 in the case of normal saline. The pres-ence of the 787 cm�1 peak observed for the RBC diluted withnormal saline also ascertains the abovementioned fact.29 Thisagain corroborates with the assumption of the deoxygenatedstate transition in red blood cells in the presence of saline.

Principal component analysis (PCA) was performed usingthe GRAMS soware to obtain statistical discrimination amongthe spectral information of RBCs suspended in normal salineand plasma. This statistical analysis was carried out on a total of30 spectra, with 15 spectra each for the two sets of cells sepa-rately diluted in plasma and in saline obtained from threedifferent individuals. Fig. 4 shows the PCA results obtained fornormal saline and plasma in the region 1100–1700 cm�1, wherethe major oxygenation markers are observed. Fig. 4(a) repre-sents the plot between the sample number and the score offactor 1. It is evident from the plot that all the normal salinesamples have negative scores, and the plasma-incubatedsamples have positive scores. The plot between the scores offactor one and the scores of factor two is depicted in Fig. 4(b). Aclear distinction was observed between the Raman spectra ofthe two classes: red blood cells in normal saline as one class andplasma-diluted samples as the other. Most of the RBCs innormal saline lie in the rst and second quadrants, whereas allthe plasma samples appeared in the third and fourthquadrants.

As is well-known, more than 90% of hemoglobin is oxygen-ated during the time of blood donation.30 However, in thepresent case, the bands representing the deoxygenationmarkers displayed a remarkable intensity enhancement fornormal saline as compared to the case of blood plasma. Thisobservation is drawn by monitoring the marker peaks mainly at1209 cm�1, 1521 cm�1, 1544 cm�1 and 1602 cm�1. Thus, weobserved strong evidence for the increased deoxygenation ofhemoglobin of the red blood cells diluted with normal saline.The results obtained are in good agreement with those reportedin previous studies regarding the oxygenation states of hemo-globin.29,31 Studies have already been reported on the deoxy-genation of red blood cells via laser-induced optical trapping,mechanical stretching, etc.26,31 Although Raman tweezers arewidely used to study the oxygenation status of red blood cells,

Fig. 4 The PCA analysis plot of Raman spectra of the RBCs diluted in noand (b) scores of factor 1 vs. scores of factor 2.

7882 | RSC Adv., 2019, 9, 7878–7884

normal saline-induced deoxygenation has not been reported todate. This is the rst report describing the normal saline-induced deoxygenation in RBCs investigated using the Ramantweezers technique. Even in the recent studies on the Ramantweezers investigation of red blood cells, normal saline hasbeen utilized as media, which results in obtaining deoxygenatedspectra for control RBCs.32 This study also highlights thenecessity of performing investigations on the red blood cells inplasma to track the eventual deoxygenation of hemoglobin.

Ramanmeasurements were also performed by saturating thesample solutions (RBCs in normal saline as well as in bloodplasma) via purging medical oxygen for one hour. The experi-mental results have again ascertained the hemoglobin deoxy-genation tendency of red blood cells in normal saline. Evenaer oxygen purging, the spectra remained the same as those ofthe deoxygenated hemoglobin in the case of RBCs suspended innormal saline. Although the initial spectra obtained immedi-ately aer oxygen purging (Fig. 5) indicated a slight increase inthe oxygenation tendency, this instant trend was found to beunstable, and it did not sustain in normal saline for more time.It is clearly evident from the switching back of the ratio1209 cm�1/1222 cm�1 (peaks in the methine deformation

rmal saline and blood plasma: (a) sample number vs. scores of factor 1

This journal is © The Royal Society of Chemistry 2019

Fig. 7 Power-dependent Raman spectra of RBC in blood plasma.

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region) and the reversal in the intensity of the oxygenationmarker at 1639 cm�1 (Fig. 5) that saline induces and maintainsthe deoxygenation trend in red blood cells. In the case of bloodplasma, oxygenation increases during oxygen purging; this canbe clearly observed from the methine deformation regionshown in Fig. 6. The oxygenation trend is stable andmaintainedin the case of blood plasma.

The power levels that are used to obtain the Raman spectraof the control (blood plasma) and normal saline-incubated redblood cells are well below the threshold for the occurrence oflaser-induced deoxygenation of RBCs. The dependence of poweron the hemoglobin oxygenation states for blood plasma wasinvestigated. As shown in Fig. 7, spectra were obtained withdifferent laser powers for the optically trapped RBCs suspendedin blood plasma. It is clear from the Raman spectra thatsignicant changes in the hemoglobin oxygenation states areobserved only aer 14 mW. There were no obvious changes tillthe laser power reached 10 mW. Thus, it is clearly evident fromthe results that the variations, including intensity ipping of themethine deformation peaks at 1209 cm�1 and 1222 cm�1, arenot photo-induced due to the presence of the 785 nm laserbeam. Hence, the spectral changes presented in Fig. 2 and 3 areentirely ascribable to the saline-induced changes in the hemeconformations.

Transfusion of the packed red cell concentrate (PRBC) tocritically ill patients plays a vital role in maintaining the oxygensaturation levels of tissues, thereby preventing hypoxia-inducedorgan damage. Most of the times, patients in the intensive careunits (ICU) have an increased demand of PRBC transfusionsbecause of their clinical conditions and low hemoglobin levels.These patients will also be receiving normal saline as intravenous uid therapy to support their intravascular volumestatus. Apart from the use of normal saline as IV uids, washingof red cells is conducted with normal saline, which can alter thePRBCs' oxygen-carrying capacity. The in vivo effect of normalsaline on the oxygen-carrying capacity has to be consideredbecause the current study clearly shows that the hemoglobinmolecule changes its conformation when it is mixed withnormal saline ex vivo.

Fig. 6 Oxygen purging of RBC in plasma.

This journal is © The Royal Society of Chemistry 2019

Conclusions

Herein, Raman tweezers spectroscopy was explored to investi-gate the oxygenation state transitions of red blood cells inplasma and 0.9% normal saline. This, in turn, could be detectedby monitoring the wavenumber shis associated with theRaman marker bands for the R (relaxed) to the T (tensed)transitions. From the spectral comparison and assessment, thestudy concluded the transformation of hemoglobin from theoxy state to deoxy state when the cell mediumwas changed fromblood plasma to normal saline. In addition, the principalcomponent analysis supported the spectral variations obtainedvia the Raman studies by displaying signicant differentiationamong the red blood cells suspended in normal saline andthose suspended in blood plasma. The present results demandmore investigations on the evaluation of the impact of saline-induced deoxygenation on red blood cells, especially inmedical settings.

Conflicts of interest

There are no conicts to declare.

Acknowledgements

Authors are thankful to DBT, DST, DST-FIST, Govt. of India andVGST, Govt. of Karnataka for providing the nancial support forthe fellows working in the project. Jijo Lukose also acknowledgeManipal Academy of Higher Education for the post doctoralfellowship. Authors also like to acknowledge Dr Sajan D. Georgefor his valuable suggestions, Vittal Shenoy, Mahendra Acharyaand the staffs in Blood Bank-KMC, Manipal for their supportduring this research work.

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